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ISAC's perspective on data: standards, structure and analysis

J. Paul RobinsonSVM Professor of Cytomics

Purdue UniversityPresident, International Society for Analytical Cytology

(ISAC)

The HTS rationale• The infinite monkey theorem defines the HTS rationale. It

states that a monkey hitting keys at random on a typewriter keyboard will almost surely eventually type every book in France's Bibliothèque Nationale de France (National Library).

• In the restatement of the theorem most popular among English speakers, the monkeys eventually type out the collected works of William Shakespeare.

• The original image was presented in Émile Borel's 1913 book "Mécanique Statistique et Irréversibilité”.

http://forum.swarthmore.edu/dr.math/problems/bridge8.5.98.htmlhttp://www.nutters.org/monkeys.html

One perspective…

BioIT Magazine2002

How many standards can you afford?

SssssssssSsssSssSssSssss

Monkey Business

• Let’s look at the infinite monkey theorem again…

• In 2003, scientists at Paignton Zoo and the University of Plymouth, in Devon in England reported that they had left a computer keyboard in the enclosure of six Sulawesi Crested Macaques for a month

• Not only did the monkeys produce nothing but five pages consisting largely of the letter S, they started by attacking the keyboard with a stone, and continued by urinating and defecating on it.

Historical Picture

• Began with flow cytometry– Invented in 1960s– 1970s started with single fluorescence signal and two

laser scatter signals – total 3 variables – 1977 Herzenberg et al 2 color flow compensation– 1980s two fluorescence signals and two scatter

signals– 1990s three to 11 fluorescence signals and two

scatter signals– 2000s 32 fluorescence signals and 10-15 scatter

signals

Fundamentals of Flow Cytometry

PMT 1

PMT 2

PMT 5

PMT 4

DichroicFilters

BandpassFilters

Laser(s)

Flow chamber

PMT 3

Scatter

Sensor

Sample

103 to 105 cells/sec

4-50 variables/cell

Advanced polychromatic cytometry

From Mario Roederer

ISAC Standards Implementation1984 – Introduction of FCS 1.0 (Flow Cytometry Standard)

- Murphy & Chused

1989 - FCS 2.0

1997 - FCS 3.0

• Cytometry 28:118-122 (1997)

2008 - FCS 4.0Proposed major update

Main challenges

• HCS activities must leverage information sciences – integrating information from genomics, emerging protein interaction networks, and ongoing chemical-genetic studies into a public knowledge base of biological systems

• Standards have to be defined and followed!

What is the issue?• There are thousands of analytical and diagnostic instrument in

clinics and labs• These instruments are manufactured by 10-20 different

companies• They use reagents from 1-200 companies for the same tests• Many tests used fluorescence as the reporter system• Imaging systems are not uniform – there are no accepted

imaging standards• There are no algorithm evaluation standards• There is no way to take data sets into any single management

engine• There are no organized databases that can evaluate the vast

amount of research or clinical data collected

How Big a Problem is it?

• 20,000 flow cytometers - 10 manufacturers• 5-8,000 confocal microscopes - 10 manufacturers• 20-50,000 other fluorescence systems – 20 manufacturers• 5-10,000 DNA microarray readers – 10 manufacturers• 500 Laser Scanning Microscopes – 2 manufacturers• 1000 plus HCS instruments – 20 manufacturers

How much data is enough? Flow assay

• Standard 7 tube assay• Each tube 7 colors plus 2 scatter parameters• 50,000 cells per tube• 450,000 parameters per tube x 7 =3,150,000• Run 25 patients/tests per day= 25x7=175

assays= 551,250,000 points per day• If you ran this assay 100 times in a year you

would have 55,125,000,000 points

How much data is enough? HCS assay

• 384 well plate assay• 6 images per well = 2304 per plate• Each image contains 100 cells (450k per image 600x800

pixels) (1,036,800k of image space = 1 Gbyte)• We collect 20 parameters per cell• We have 230,400 x 20 parameters = 4,608,000• For a 10 plate assay we have 46,080,000 parameters• If we run 2 assays a day 5 days a week for 40 weeks• 46,080,000 x 2 x 5 x 40 = 18,432,000,000 parameters• Total storage space =1 Gbyte x 10 x 2 x 5 x 40 = 4 Tbytes

How many images are there…

• Industry estimates indicate that 80 billion new images are created every year

• 219,178,082 per day• 9,132,420 per hour• 152,207 per minute• 2,536 per second

http://a06.cgpublisher.com/proposals/244/index_html

Calibration and Standards?• Very few real standards• Local calibration if at all• Standards processes must be created and implemented

across several fields• Necessary to identify

– Instrument standards– Reagent Standards– Analysis Standards– Data structure standards– Metadata standards– Algorithm identification (at least)

Identical microbeads with various calibrated binding capacities of goat-anti-mouse IgG on their surface:

Eve

nts

Mean fluorescence intensity (MFI)

1 2

3 4

Blank

QSC, Cat. No. 815Bangs Laboratories, Inc.www.bangslabs.com

Antibody binding capacity (ABC) provided by the manufacturer :

Blank. 0 MESF

1. 6851 MESF

2. 23379 MESF

3. 58333 MESF

4. 213369 MESF

bead

Ab site

MESF=Molecules of equivalent soluble fluorochrome

QSC Beads (Quantum Simply Cellular)

Noise measurement with a standard

R. M. Zucker and O. Price, Cytometry 43 (2001) 273 - 294

QC- Optical Filters

• Depending on location, filters can be placed under extreme stress• Environmental conditions (humidity)

Excitation Efficiency Profiles

(note – there really isn’t a 545 nm line available!!)

Management compensation of fluorescence overlap becomes crucial

Noise measurement in the images II

Wavelet

transform 1

Wavelet

transform 2

signal

wavelet shrinkage

effect ?

noise

YES

NO

scale corr. s/n filter

s/n childs/n

parent

raw image

denoised image (signal)

diff.

Light detector stability analysis I

ttt

ststt

ebI

bb

trend component: random walks (RW)

periodic component: dynamic harmonic regression

j

tjjtjjtt e)}tsin(b)tcos(a{I

What Standards are available?

• Beads for size, intensity, color• No calibration tools available for high

resolution optical microscope (Richardson slide no longer manufactured)

• 1990 we created the Handbook of Flow Cytometry Methods to exactly define methods

• 1997 we created Current Protocols in Cytometry

About original data…“It is crucially important to keep your original digital or analog data exactly asthey were acquired and to record yourinstrument settings. This primary ruleof good scientific practice will allowyou or others to return to your originaldata to see whether any informationwas lost by the adjustments made tothe images. In fact, some journal reviewersor editors request access to suchprimary data to ensure accuracy.”

J Cell Biol. 166:11-15, 2004

Workshop on Standards and Calibration in Cytometry and Biological Imaging Modalities

Jointly sponsored by

International Society for Analytical Cytology (ISAC)& the Society for Biomolecular Sciences (SBS)

Site and date not yet set

To highlight the areas of cell analysis that need to be standardized To develop a series of recommendations on:

– Data file standards– Imaging standards– Archival/storage standards– Compression modalities– Algorithms and processing – Analytical technologies

ISAC 21st Century

• Flow and imaging are equally emphasized in ISAC

• Standards and Calibration

• Biosafety issue

• Core managers support

• Education

• Public Policy

• www.isac-net.org

• www.cyto.purdue.edu– Cytometry web/email discussion– Educational materials, Tutorials, Lectures

Next ISAC Congress May 17-21, 2008, Budapest, Hungary

Some ReferencesR.A. Hoffman, Current Protocols in Cytometry, 1997 : 1.3.1-1.3.19J.C.S. Wood, Current Protocols in Cytometry, 1997 : 1.4.1-1.4.12Cytometry, Volume 33, Number 2, 1998R. M. Zucker and O. Price, Cytometry 43 (2001) 273 - 294